FPOP

Although protein footprinting results are commonly obtained by ESI-based LC-MS/MS, a more rapid-turnaround alternative approach is desirable to expand the scope of protein footprinting and facilitate routine analysis such as monitoring protein high order structure in quality control or checking epitope maps. Considering that MALDI is a faster procedure that can be easily adapted for high-throughput analysis, we explore here the feasibility of developing a MALDI-based analysis "portfolio" of bottom-up peptide mass mapping for footprinting...

Protein radical labeling, like fast photochemical oxidation of proteins (FPOP), coupled to a top-down mass spectrometry (MS) analysis offers an alternative analytical method for probing protein structure or protein interaction with other biomolecules, for instance, proteins and DNA. However, with the increasing mass of studied analytes, the MS/MS spectra become complex and exhibit a low signal-to-noise ratio. Nevertheless, these difficulties may be overcome by protein isotope depletion. Thus, we aimed to use protein isotope depletion to analyze FPOP-oxidized samples by top-down MS analysis...

Fast Photochemical Oxidation of Proteins (FPOP) is a promising technique for studying protein structure and dynamics. The quality of insight provided by FPOP depends on the reliability of the determination of the modification site. This study investigates the performance of two search engines, Mascot and PEAKS, for the data processing of FPOP analyses. Comparison of Mascot and PEAKS of the hemoglobin--haptoglobin Bruker timsTOF data set (PXD021621) revealed greater consistency in the Mascot identification of modified peptides, with around 26% of the IDs being mutual for all three replicates, compared to approximately 22% for PEAKS...

Characterizing changes in the higher order structure (HOS) of monoclonal antibodies upon stressed conditions is critical to gaining a better understanding of the product and process. One single biophysical approach may not be best suited to assess HOS comprehensively; thus, the synergy from multiple, complementary approaches improves characterization accuracy and resolution. In this study, we employed two mass spectrometry (MS )-based footprinting techniques, namely, fast photochemical oxidation of proteins (FPOP)-MS and hydrogen-deuterium exchange (HDX)-MS, supported by dynamic light scattering (DLS), differential scanning calorimetry (DSC), circular dichroism (CD), and nuclear magnetic resonance (NMR) to study changes to the HOS of a mAb upon thermal stress...

Monitoring protein structure before and after environmental alterations (e.g., different cell states) can give insights into the role and function of proteins. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry (MS) allows for monitoring of structural rearrangements by exposing proteins to OH radicals that oxidize solvent-accessible residues, indicating protein regions undergoing movement. Some of the benefits of FPOP include high throughput and a lack of scrambling due to label irreversibility...

Irreversible protein footprinting is a mass spectrometry-based approach in which solvent-accessible sites of a protein are modified to assess high-order protein structure. Structural insights can be gained by determining the position and extents of modification. The usual approach to obtain the "footprint" is to analyze the protein through bottom-up LC-MS/MS. In this approach, the proteins are digested to yield a mixture of peptides that are then separated by LC before locating the modification sites by MS/MS...

INTRODUCTION: Lifetime risk of surgery for female pelvic organ prolapse (FPOP) is estimated at 10 to 20%. Prolapse assessment is mostly done by clinical examination. Perineal ultrasound is easily available and performed to evaluate and stage FPOP. This study's aim is to evaluate the agreement between clinical examination by POP-Q and perineal sonography in women presenting pelvic organ prolapse. MATERIALS AND METHODS: We carried out a prospective study from December 2015 to March 2018 in the gynecologic department of a teaching hospital...

Monitoring protein structure before and after perturbations can give insights into the role and function of proteins. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry (MS) allows monitoring of structural rearrangements by exposing proteins to OH radicals that oxidize solvent accessible residues, indicating protein regions undergoing movement. Some of the benefits of FPOP include high throughput and lack of scrambling due to label irreversibility. However, the challenges of processing FPOP data have thus far limited its proteome-scale uses...

Understanding the higher-order structure of membrane proteins (MPs), which are vital for numerous biological processes, is crucial for comprehending their function. Although several biophysical approaches have been used to study the structure of MPs, limitations exist owing to the proteins' dynamic nature and heterogeneity. Mass spectrometry (MS) is emerging as a powerful tool for investigating membrane protein structure and dynamics. Studying MPs using MS, however, must meet several challenges including the lack of stability and solubility of MPs, the complexity of the protein-membrane system, and the difficulty of digestion and detection...

Daptomycin is a cyclic lipopeptide antibiotic that targets the lipid membrane of Gram-positive bacteria. Membrane fluidity and charge can affect daptomycin activity, but its mechanisms are poorly understood because it is challenging to study daptomycin interactions within lipid bilayers. Here, we combined native mass spectrometry (MS) and fast photochemical oxidation of peptides (FPOP) to study daptomycin-membrane interactions with different lipid bilayer nanodiscs. Native MS suggests that daptomycin incorporates randomly and does not prefer any specific oligomeric states when integrated into bilayers...

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Many cancer drugs fail at treating solid epithelial tumors with hypoxia and insufficient drug penetration thought to be contributing factors to the observed chemoresistance. Owing to this, it is imperative to evaluate potential cancer drugs in conditions as close to in vivo as possible, which is not always done. To address this, we developed a mass spectrometry-based protein footprinting method for exploring the impact of hypoxia on protein in 3D colorectal cancer cells. Our group has previously extended the protein footprinting method fast photochemical oxidation of proteins (FPOP) for live cell analysis (IC-FPOP); however, this is the first application of IC-FPOP in a 3D cancer model...

Commercial monoclonal antibodies are growing and important components of modern therapies against a multitude of human diseases. Well-known high-resolution structural methods such as protein crystallography are often used to characterize antibody structures and to determine paratope and/or epitope binding regions in order to refine antibody design. However, many standard structural techniques require specialized sample preparation that may perturb antibody structure or require high concentrations or other conditions that are far from the conditions conducive to the accurate determination of antigen binding or kinetics...

Detection of host cell protein (HCP) impurities is critical to ensuring that recombinant drug products, including monoclonal antibodies (mAbs), are safe. Mechanistic characterization as to how HCPs persist in drug products is important to refining downstream processing. It has been hypothesized that weak lipase-mAb interactions enable HCP lipases to evade drug purification processes. Here, we apply state-of-the-art methods to establish lipase-mAb binding mechanisms. First, the mass spectrometry (MS) approach of fast photochemical oxidation of proteins was used to elucidate putative binding regions...

Amyloid fibrils, insoluble β-sheets structures that arise from protein misfolding, are associated with several neurodegenerative disorders. Many small molecules have been investigated to prevent amyloid fibrils from forming; however, there are currently no therapeutics to combat these diseases. Mass spectrometry (MS) is proving to be effective for studying the high order structure (HOS) of aggregating proteins and for determining structural changes accompanying protein-inhibitor interactions. When combined with native MS (nMS), gas-phase ion mobility, protein footprinting, and chemical cross-linking, MS can afford regional and sometimes amino acid spatial resolution of the aggregating protein...

A fluorescent porous organic polymer (FPOP) with strong fluorescence and tunable emission colors, was synthesized through a simple cost-effective method via Scholl coupling reaction. Experiments proved the stability and excellent detection and adsorption ability, and microporous nature of the material. Luminescence of FPOP was quenched when addition of nitroaromatic compounds. The properties along with large-scale and low-cost preparation make these FPOP potential candidates for fluorescence detection of nitroaromatic compounds...

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical footprinting approach whereby radicals, produced by UV laser photolysis of hydrogen peroxide, induce oxidation of amino acid side-chains. Mass Spectrometry (MS) is employed to locate and quantify the resulting irreversible, covalent oxidations to use as a surrogate for side-chain solvent accessibility. Modulation of oxidation levels under different conditions allows for the characterisation of protein conformation, dynamics and binding epitopes...

Fast photochemical oxidation of proteins (FPOP) footprinting is a structural mass spectrometry method that maps proteins by fast and irreversible chemical reactions. The position of oxidative modification reflects solvent accessibility and site reactivity and thus provides information about protein conformation, structural dynamics, and interactions. Bottom-up mass spectrometry is an established standard method to analyze FPOP samples. In the bottom-up approach, all forms of the protein are digested together by a protease of choice, which results in a mixture of peptides from various subpopulations of proteins with varying degrees of photochemical oxidation...

Monoclonal antibody (mAb) has grown to be the major asset in protein therapeutics market since its initial introduction in 1980s. To identify a suitable mAb as a drug candidate for development requires deep understanding of disease biology and attribute sciences, including epitope-paratope binding recognition. Mass spectrometry (MS) has become a critical technology platform in epitope mapping. MS-based approaches utilize chemical labeling to assess the changes of solvent accessibilities during binding interactions, and the labeling can be either reversible or irreversible...

A combination of covalent labeling techniques and mass spectrometry (MS) is currently a progressive approach for deriving insights related to the mapping of protein surfaces or protein-ligand interactions. In this study, we mapped an interaction interface between the DNA binding domain (DBD) of FOXO4 protein and the DNA binding element (DAF16) using fast photochemical oxidation of proteins (FPOP). Residues involved in protein-DNA interaction were identified using the bottom-up approach. To confirm the findings and avoid a misinterpretation of the obtained data, caused by possible multiple radical oxidations leading to the protein surface alteration and oxidation of deeply buried amino acid residues, a top-down approach was employed for the first time in FPOP analysis...